Highly reproducible agglutination immunoassay method and reagents

ABSTRACT

The present invention provides an agglutination immunoassay, wherein the agglutination of insoluble carrier particles such as latex are stabilized and uniformized to give good reproducibility, and a reagent therefor. In the agglutination immunoassay which comprises allowing an antigenic substance in a sample to bind to insoluble carrier particles carrying substantially neither antigens nor antibodies thereon, and allowing an antibody or an antibody complex which reacts specifically to the antigenic substance to bind to the antigenic substance to give a selective agglutination of the insoluble carrier particles, a homopolymer prepared by polymerization of a monomer such as 2-methacryloyloxyethyl phosphorylcholine having a phosphorylcholine group and a vinyl group, or a copolymer prepared by polymerization of a monomer having a phosphorylcholine group and a vinyl group, with a monomer having a vinyl group such as n-butyl methacrylate is used.

TECHNICAL FIELD

[0001] The present invention relates to an agglutination immunoassay,wherein an antigenic substance in an aqueous medium such as a livingsample is determined immunologically by an agglutination reaction, and areagent therefor.

BACKGROUND ART

[0002] Recently, automation of various examination such as laboratorytests and reduction of time for determination are attempted inhospitals, inspection institutions and the like in the light of shortageof manpower, cost reduction, a large quantity of samples to be treated,and the like. As a method suitable for such automation, attentions aredrawn to an agglutination immunoassay, wherein an antigenic substance isdetermined by an agglutination reaction of insoluble carrier particles.For example, Japanese Published Unexamined Patent Application No.35752/95 discloses an agglutination immunoassay which comprises allowingan antigenic substance in a sample to bind to insoluble carrierparticles carrying substantially neither antigens nor antibodiesthereon, and allowing an antibody or an antibody complex which reactsspecifically to the antigenic substance to bind to the antigenicsubstance to give a selective agglutination of the insoluble carrierparticles.

[0003] However, in an agglutination immunoassay, it is difficult to havea reaction with good reproducibility due to the non-uniformity ofagglutination in an agglutination reaction of the insoluble carrierparticles with antigens or antibodies. Specifically, in an agglutinationimmunoassay which comprises allowing an antigenic substance in a sampleto bind to insoluble carrier particles carrying substantially neitherantigens nor antibodies thereon, and allowing an antibody or an antibodycomplex which reacts specifically to the antigenic substance to bind tothe antigenic substance to give a selective agglutination of theinsoluble carrier particles, there are problems such as direct bindingof antibodies to the insoluble carrier particles. Therefore, improvementof reproducibility in the agglutination immunoassay is required.

[0004] An object of the present invention is to provide: anagglutination immunoassay which comprises allowing an antigenicsubstance in a sample to bind to insoluble carrier particles carryingsubstantially neither antigens nor antibodies thereon, and allowing anantibody or an antibody complex which reacts specifically to theantigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles due tostabilization and uniformization of an agglutination as well asprevention of non-specific adsorption; and a reagent therefor.

DISCLOSURE OF THE INVENTION

[0005] The present inventors have made a keen study to solve theabove-mentioned problem and have found out that in an agglutinationimmunoassay which comprises allowing an antigenic substance in a sampleto bind to insoluble carrier particles carrying substantially neitherantigens nor antibodies thereon, and allowing an antibody or an antibodycomplex which reacts specifically to the antigenic substance to bind tothe antigenic substance to give a selective agglutination of theinsoluble carrier particles, a compound having a group analogous to aphosphorylcholine group has an action to promote and uniformize theagglutination of the carrier particles by an antigen-antibody reactionto give a stable and uniform agglutination as well as improvement inreproducibility. The present invention has been thus completed.

[0006] The present invention relates to an agglutination immunoassaywhich comprises allowing an antigenic substance in a sample to bind toinsoluble carrier particles carrying substantially neither antigens norantibodies thereon, and allowing an antibody or an antibody complexwhich reacts specifically to the antigenic substance to bind to theantigenic substance to give a selective agglutination of the insolublecarrier particles, wherein a compound having a group represented byformula (I) (wherein n is an integer of 1 to 6,and R¹, R² and R³ are thesame or different, and independently represent hydrogen, substituted orunsubsitituted alkyl having 1 to 6 carbon atoms) is used (claim 1); theagglutination immunoassay according to claim 1, wherein the compoundhaving a group represented by formula (I) is a compound prepared bypolymerization of a monomer having a group represented by formula (I)(claim 2); the agglutination immunoassay according to claim 1, whereinthe compound having a group represented by formula (I) is a compoundprepared by polymerization of a monomer having a group represented byformula (I) with other monomer polymerizable with the monomer having agroup represented by formula (I) (claim 3); the agglutinationimmunoassay according to claim 3, wherein the other monomerpolymerizable with the monomer having a group represented by formula (I)is a monomer having a vinyl group (claim 4); the agglutinationimmunoassay according to claim 4, wherein the monomer having a vinylgroup is n-butyl methacrylate (claim 5); the agglutination immunoassayaccording to any of claims 2 to 5, wherein the monomer having a grouprepresented by formula (I) is a monomer having a group represented byformula (I) and a vinyl group (claim 6); the agglutination immunoassayaccording to any of claims 1 to 6, wherein the group represented byformula (I) is a phosphorylcholine group (claim 7); the agglutinationimmunoassay according to claim 6, wherein the monomer having a grouprepresented by formula (I) and a vinyl group is 2-methacryloyloxyethylphosphorylcholine (claim 8); the agglutination immunoassay according toany of claims 1 to 8, wherein the antibody is an anti-hemoglobin A1cmonoclonal antibody (claim 9); the agglutination immunoassay accordingto any of claims 1 to 9, wherein the antibody complex comprises anantibody which reacts specifically to the antigenic substance, and asecondary antibody which reacts selectively to the antibody which reactsspecifically to the antigenic substance (claim 10); and theagglutination immunoassay according to any of claims 1 to 10, whereinthe insoluble carrier particles are polystyrene latex (claim 11).

[0007] Furthermore, the present invention relates to a reagent for animmunoassay comprising a compound having a group represented by formula(I) (wherein n is an integer of 1 to 6, and R¹, R² and R³ are the sameor different, and independently represent hydrogen, substituted orunsubsitituted alkyl having 1 to 6 carbon atoms) used in anagglutination immunoassay which comprises allowing an antigenicsubstance in a sample to bind to insoluble carrier particles carryingsubstantially neither antigens nor antibodies thereon, and allowing anantibody or an antibody complex which reacts specifically to theantigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles (claim 12);the reagent for an immunoassay according to claim 12, wherein thecompound having a group represented by formula (I) is a compoundprepared by polymerization of a monomer having a group represented byformula (I) (claim 13); the reagent for an immunoassay according toclaim 12, wherein the compound having a group represented by formula (I)is a compound prepared by polymerization of a monomer having a grouprepresented by formula (I) with other monomer polymerizable with themonomer having a group represented by formula (I) (claim 14); thereagent for an immunoassay according to claim 14, wherein the othermonomer polymerizable with the monomer having a group represented byformula (I) is a monomer having a vinyl group (claim 15); the reagentfor an immunoassay according to claim 15, wherein the monomer having avinyl group is n-butyl methacrylate (claim 16); the reagent for animmunoassay according to any of claims 13 to 16, wherein the monomerhaving a group represented by formula (I) is a monomer having a grouprepresented by formula (I) and a vinyl group (claim 17); the reagent foran immunoassay according to any of claims 12 to 17, wherein the grouprepresented by formula (I) is a phosphorylcholine group (claim 18); thereagent for an immunoassay according to claim 17, wherein the monomerhaving a group represented by formula (I) and a vinyl group is2-methacryloyloxyehtyl phosphorylcholine (claim 19); the reagent for animmunoassay according to any of claims 12 to 19, wherein the antibody isan anti-hemoglobin A1c monoclonal antibody (claim 20); the reagent foran immunoassay according to any of claims 12 to 20, wherein the antibodycomplex comprises an antibody which reacts specifically to the antigenicsubstance, and a secondary antibody which reacts selectively to theantibody which reacts specifically to the antigenic substance (claim21); and the reagent for an immunoassay according to any of claims 12 to21, wherein the insoluble carrier particles are polystyrene latex (claim22).

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] There is no specific limitation to an agglutination immunoassayaccording to the present invention as long as the agglutinationimmunoassay is an immunoassay which comprises allowing an antigenicsubstance in a sample to bind to insoluble carrier particles carryingsubstantially neither antigens nor antibodies thereon, and allowing anantibody or an antibody complex which reacts specifically to theantigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles, wherein acompound having a group represented by formula (I), shown below, whereinn is an integer of 1 to 6,and R¹, R² and R³ are the same or different,and independently represent hydrogen, substituted or unsubsititutedalkyl having 1 to 6 carbon atoms, is used. Further, there is no specificlimitation to a reagent for an immunoassay according to the presentinvention as long as the reagent comprises a compound having a groupanalogous to a phosphorylcholine group represented by formula (I), andis used in an agglutination immunoassay which comprises allowing anantigenic substance in a sample to bind to insoluble carrier particlescarrying substantially neither antigens nor antibodies thereon, andallowing an antibody or an antibody complex which reacts specifically tothe antigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles. The bindingherein refers to both physical adsorption and chemical bond.

[0009] There is no specific limitation to insoluble carrier particle forthe present invention as long as the insoluble carrier particlescarrying substantially neither antigens nor antibodies thereon arecapable of binding an antigenic substance in a sample. The examplesinclude the known microparticles of an organic polymer described inJapanese Published Examined Patent Application No. 11575/83,microparticles of inorganic oxides or microparticles wherein the surfaceof these substances that are to form the core is treated with an organicsubstance or the like. The specific examples are synthetic resin (latex)such as polystyrene, polyvinylchloride, polypropylene, (meth)acrylicresin and poly(methyl methacrylate); cellulose derivatives such asnitrocellulose, cellulose and methylcellulose; and inorganic substancessuch as metal, ceramics, glass and silicon rubber. Among thesesubstances, a polystyrene synthetic polymer, particularly a polystyrenesynthetic polymer co-polymerized with an acrylate monomer or a monomerhaving sulfonic acid as a component to provide electric charges ispreferable.

[0010] As described above, latex particles, in particular, such aspolystyrene latex are preferably used in the present invention asinsoluble carrier particles. Proteins and peptides can smoothly beadsorbed on latex having a surface of high hydrophobicity, such aspolystyrene latex. In addition, polystyrene particles prepared by asoap-free polymerization, may preferably be used particularly, becausethey can remain stable without surfactants due to the repulsion raisedbetween negative charges on the surface. Alternatively, various kinds ofdenatured latex (for example, denatured carboxylic acid latex), magneticlatex (latex containing magnetic particles) and the like may also beused, if necessary.

[0011] As to insoluble carrier particles, equality of the size,regulation of the surface condition, selection of the internal structureand so on are usually required at a high level for conductingquantitative immunoassay, and insoluble carrier particles such as latexfavorable for the preparation of the reagents can be selected from thosecommercially available. A shape of insoluble carrier particles is notparticularly limited, and a sphere shape is exemplified. As a particlediameter in sphere-shaped particles, for instance, 0.03 to 0.8 μm onaverage is preferable, and 0.06 to 0.2 μm on average is more preferable.In the present invention, there is no specific limitation to aconcentration of insoluble carrier particles in a reaction solution, andthe concentration is, for example, 0.001 to 10% by weight, preferably0.005 to 5% by weight and more preferably 0.01 to 2% by weight tostabilize and uniformize the agglutination reaction of insoluble carrierparticles more.

[0012] As a compound having a group represented by formula (I) in thepresent invention, there is no specific limitation as long as thecompound is a compound having a group represented by formula (I) whereinn is an integer of 1 to 6,and R¹, R² and R³may be the same or different,and independently represent hydrogen, substituted or unsubstituted alkylhaving 1 to 6 carbon atoms. Examples of alkyl part of R¹, R² and R³ informula (I) include methyl, ethyl, propyl, butyl, isobutyl, pentyl,hexyl and the like. Moreover, number of substituents in the substitutedalkyl is 1 to 3, and examples of the substituent include hydroxy, aryland the like. Examples of the aryl include benzyl, naphthyl and thelike.

[0013] In addition, examples of the compound having a group representedby formula (I) include a compound prepared by polymerization of amonomer having a group represented by formula (I), a compound preparedby copolymerization of a monomer having a group represented by formula(I) with other monomer polymerizable with the monomer having a grouprepresented by formula (I), and the like. As the monomer having a grouprepresented by formula (I), a monomer having a group represented byformula (I) and a vinyl group is preferably exemplified. Furthermore, asother monomer polymerizable with the monomer having a group representedby formula (I), a monomer having a vinyl group is preferablyexemplified.

[0014] As a group represented by formula (I) in the present invention, aphosphorylcholine group (hereinafter abbreviated as PC group) ispreferably exemplified. Therefore, as a compound having a grouprepresented by formula (I) in this present invention, a compound havingPC group is preferably exemplified. In addtion, there is no specificlimitation to a compound having PC group. As a compound having a PCgroup, a polymer prepared by polymerization of a monomer having a PCgroup, and a polymer prepared by copolymerization of a monomer having aPC group with other monomer polymerizable with the monomer having a PCgroup are preferably exemplified. Among the compound having a PC group,a polymer prepared by polymerization of a monomer having a PC group anda vinyl group, and a polymer prepared by copolymerization of a monomerhaving a PC group and a vinyl group with other monomer polymerizablewith the monomer having a PC group and a vinyl group are more preferablyexemplified.

[0015] There is no specific limitation to a monomer having a PC groupand a vinyl group. Examples of the monomer having a PC group and a vinylgroup include 2-acryloyloxyethyl phosphorylcholine,2-methacryloyloxyethyl phosphorylcholine (abbreviated hereinafter asMPC), 2-(meth)acryloyloxyethoxyethyl phosphorylcholine,6-(meth)acryloyloxyhexyl phosphorylcholine,10-(meth)acryloyloxyethoxynonyl phosphorylcholine, allylphosphorylcholine, butenyl phosphorylcholine, hexenyl phosphorylcholine,octenyl phosphorylcholine, decenyl phosphorylcholine, and the like. Inaddition, these monomers can be prepared according to the known methodsdescribed in Japanese Published Unexamined Patent Application No.6325/79, Japanese Published Unexamined Patent Application No. 154591/83,and the like.

[0016] Other monomers polymerizable with the monomer having a PC group,preferably the monomers having a vinyl group include a (meth)acrylatesuch as methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl(meth)acrylate, isobutyl (meth)acrylate, pentyl (meth)acrylate, hexyl(meth)acrylate, heptyl (meth)acrylate, octyl (meth)acrylate, tridecyl(meth)acrylate, 2-hydroxyethyl methacrylate; a styrene monomer such asstyrene, α-methyl styrene, styrene having a phenyl group substitutedwith methyl group(s) and styrene having a phenyl group substituted withchlorine atom(s); a substituted or unsubstituted hydrocarbon monomersuch as vinyl chloride, vinylidene chloride, ethylene, proplylene andisobutylene; a vinylester monomer such as vinyl acetate and vinylpropionate; a vinyl ether monomer such as ethyl vinyl ether and n-butylvinyl ether; diethyl itaconate, di(n-butyl) itaconate, and the like.Among these monomers, esters of methacrylic acid (methacrylate), styreneand the like are preferable, and n-butyl methacrylate (abbreviatedhereinafter as BMA) is particularly preferable.

[0017] A polymer comprising a PC group can be prepared by a conventionalpolymerization method such as radical polymerization, wherein componentsfor polymerization comprising the above-mentioned monomer having a PCgroup are polymerized in the presence of a polymerization initiator. Asa polymerization initiator, there is no specific limitation as long asthe initiator is an initiator usually used for radical polymerization,and the following are preferably exemplified: 2,2′-azobis(2-methylpropionamidine) dihydrochloride, 4,4′-azobis(4-cyanovaleric acid),2,2′-azobis[2-(5-methyl-2-imidazoline-2-yl)propane] dihydrochloride,2,2′-azobisisobutylamide dihydrate, 2,2′-azobisisobutyronitrile,ammonium persulfate, potassium persulfate, benzoyl peroxide,diisopropylperoxy dicarbonate, t-butylperoxy 2-ethylhexanoate,t-butylperoxy pivalate, t-butylperoxy isobutylate, lauroyl peroxide,azobisisobutyronitril, 2,2′-azobis(2,4-dimethylvaleronitrile),t-butylperoxy neodecanoate, mixtures thereof, and the like. Inparticular, in case of homopolymerization of MPC or copolymerization ofMPC with other monomer such as BMA having a vinyl group,2,2′-azobisisobutyronitrile (abbreviated hereinafter as AIBN) ispreferably used as a polymerization initiator in the light ofpolymerizability.

[0018] There is no specific limitation to an amount of a polymerizationinitiator, and for 100 parts by weight of all monomers used inpolymerization, 0.01 to 10 parts by weight is preferable, and 0.1 to 5parts by weight is more preferable. Moreover, polymerization ispreferably carried out at 30 to 80. degree. C., more preferably at 40 to70. degree. C. for 2 to 72 hours. In polymerization, a solvent can beused for effective polymerization reaction. Examples of the solventinclude water, methanol, ethanol, propanol, t-butanol, benzene, toluene,dimethyl formamide, tetrahydrofuran, chloroform, a mixtures thereof, andthe like. In particular, in case of homopolymerization of MPC orcopolymerization of MPC with other monomer having a vinyl group, waterand ethanol are preferably used in the light of solubility andpolymerizability. Purification of the obtained polymers can be carriedout by general method for purification such as reprecipitation, dialysisand ultrafiltration.

[0019] As a percentage of PC group in a polymer comprising a PC group, 1to 100 mol % is preferable, and 5 to 10 mol % is more preferable perpolymer comprising a PC group. Use of the polymer comprising less than 1mol % of a PC group is not preferable because of difficulty inprevention of non-specific adsorption. In addition, average molecularweight of a polymer comprising a PC group varies depending onpolymerization temperature, amount of polymerization initiator, whethera polymerization regulating agent is used or not, and the like. Apolymer with a molecular weight (Mn) of 100 to 1,000,000 is preferable,and 1,000 to 500,000 is particularly preferable. There is no specificlimitation to concentration of a compound having a PC group in thepresent invention, and concentration of 0.0001 to 10% is preferable,concentration of 0.001 to 5% is more preferable, and concentration of0.01 to 1% is particularly preferable. Concentration less than 0.0001%is not preferable because reproducibility in determination does notimprove remarkably. Concentration more than 10% is not also preferablebecause of incorrect determination ascribed to bubbles formed byterrible bubbling. Additionally, there is no specific limitation to acompound having a PC group, but homopolymer of MPC or copolymer of MPCwith BMA is preferable.

[0020] In the present invention, agglutination immunoassay refers to anagglutination immunoassay which comprises allowing an antigenicsubstance in a sample to bind to insoluble carrier particles carryingsubstantially neither antigens nor antibodies thereon, and allowing anantibody or an antibody complex which reacts specifically to theantigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles. However, incase an antibody or an antibody complex used in the present inventionhas an effect on values to be determined by reacting not only to theantigenic substance bound to insoluble carrier particles but also to theantigenic substance not bound to insoluble carrier particles, thefollowing methods could be used as an agglutination immunoassay: amethod wherein an antigenic substance in a sample is bound to insolublecarrier particles, and the free antigenic substance is removed byrinsing the insoluble carrier particles; a method of increasing thedegrees of the antigenic substance bound to insoluble carrier particlesto the free antigenic substance; a method of using the antibody orantibody complex which reacts to the antigenic substance bound toinsoluble carrier particles, but does not substantially react to thefree antigenic substance in the liquid phase.

[0021] In the present invention, insoluble carrier particles aregenerally used as a so-called latex suspension, wherein insolublecarrier particles such as latex are suspended in an aqueous medium suchas a buffer solution. As a buffer for preparation of the buffersolution, a Good's buffer in addition to phosphoric acid buffer,carbonic acid buffer, and organic acid buffer can be used. As an acidfor adjusting the pH of a buffer solution containing the buffer, anorganic acid such as acetic acid as well as an inorganic acid such ashydrochloric acid, sulfuric acid and nitric acid can be used. Inaddition, as an alkali for adjusting the pH, sodium hydroxide, potassiumhydroxide, lithium hydroxide, ammonium hydroxide and the like can beused. Moreover, in the present invention, surfactants effective forsolubilization of lipid in a sample can also be used. In particular, anonionic surfactant having a polyoxyethyleneglycol group, a cationicsurfactant and an anionic surfactant can be used, if necessary.

[0022] As an antigenic substance in a sample which is a target to bedetermined in the agglutination immunoassay of the present invention, asubstance capable of binding to insoluble carrier particles or asubstance by which a corresponding polyclonal antibody or a monoclonalantibody can be obtained or prepared is preferable. However, in thelight of facility to prepare the polyclonal antibody or monoclonalantibody, a substance such as protein with the molecular weight of10,000 or more and glycoprotein with the molecular weight of 10,000 ormore is more preferable as an antigenic substance. As an antigenicsubstance in a sample, Hemoglobin A1c (hereinafter abbreviated as HbA1c)in a blood sample is preferably exemplified. Moreover, as an antibodyspecifically bound to an antigenic substance in the present invention, apolyclonal antibody and/or a monoclonal antibody can be used. Amonoclonal antibody can be obtained by cell fusion method reported byKoehler & Milstein (Nature, 256, 495-497, 1975). Additionally, as anantibody corresponding to the aforementioned HbA1c, an anti-HbA1cmonoclonal antibody can be exemplified.

[0023] Meanwhile, it is well known that among monoclonal antibodieswhich are produced by hybridoma cells and selected by ELISA method,there are some monoclonal antibodies that do not react in an assay suchas an radio immunoassay (RIA), wherein a reaction of an antigen with anantibody is carried out in a liquid phase. However, in the agglutinationimmunoassay according to the present invention, use of such monoclonalantibodies is preferable because reaction of the antibodies with thefree antigenic substance in the liquid phase is inhibited to give aspecific reaction of the antibodies with the antigenic substance boundto insoluble carrier particles.

[0024] In an agglutination immunoassay according to the presentinvention, there is a possibility that formation of an agglutination maynot proceed by reacting the insoluble carrier particles carrying anantigenic substance, formed by binding an antigenic substance toinsoluble carrier particles such as latex, with the monoclonal antibody.In such case, however, an agglutination can be brought about by usingthe antibody complex. That is, formation in advance of the antibodycomplex by reaction of the monoclonal antibody with the second antibodywhich reacts selectively to the monoclonal antibody, followed byreaction of the antibody complex with the antigenic substance bound toinsoluble carrier particles such as latex can bring about anagglutination of the insoluble carrier particles. In addition to theabove-mentioned method, an antibody complex can be formed by thefollowing methods: addition of avidin to a biotin-labelled antibody;chemical bond by which an antibody is bound to an enzyme to give anenzyme-labelled antibody.

[0025] According to the present invention which enables a selectiveagglutination of insoluble carrier particles by reaction of an antigenicsubstance bound to insoluble carrier particles with an antibody or anantibody complex which reacts specifically to the antigenic substance, areagent for an immunoassay can be prepared simply and easily, and areagent for an immunoassay kept highly stable during storage can besupplied. That is, as the insoluble carrier particles such as latex donot carry neither antigens nor antibodies thereon, commerciallyavailable insoluble carrier particles carrying neither antigens norantibodies thereon can be used as such. Furthermore, the antibodies usedmay not necessarily be the purified antibodies. Thus, the reagent is sosimple that the reagent can be kept stable during storage.

[0026] Further, as an aqueous medium used in an agglutinationimmunoassay which comprises allowing an antigenic substance in a sampleto bind to insoluble carrier particles carrying substantially neitherantigens nor antibodies thereon, and allowing an antibody or an antibodycomplex which reacts specifically to the antigenic substance to bind tothe antigenic substance in an aqueous medium comprising a compoundhaving a group represented by formula (I) such as phophorylcholine togive an agglutination of the insoluble carrier particles to be measured,an aqueous medium in which non-specific adsorption and binding of theantibody or the antibody complex to the insoluble carrier particles suchas latex can be prevented is preferred. Examples of the aqueous mediumin which non-specific adsorption and binding of the antibody or theantibody complex to the insoluble carrier particles can be preventedinclude an aqueous solution comprising about 0.1 to 0.3% surfactant suchas Tween 20, and the like.

[0027] There is no specific limitation to a vessel used in anagglutination reaction of the present invention, and a vessel of atubular form such as a polystyrene test tube generally used in this typeof agglutination reaction is exemplified. Moreover, in the light ofeasiness of batch treatment of many samples, a plate for ELISA havingmany wells (such as 96-well plate for ELISA “NUNC-IMMUNO PLATE”, NalgeNunc International K.K.) can be used. Additionally, in the light ofeasiness of an optical determination of agglutination of insolublecarrier particles such as latex, a reaction in a transparent vessel ispreferable. In addition, in case of determining an agglutination ofinsoluble carrier particles such as latex by an autoanalyzer,agglutination reaction is usually carried out in a reaction vessel forthe autoanalyzer.

[0028] There is no specific limitation to a method of measuring thedegrees of an agglutination of insoluble carrier particlesagglutination. In case of measuring an agglutination qualitatively orsemiquantitatively, for example, degrees of agglutination of insolublecarrier particles can be determined visually, by comparing degrees ofturbidity in measurement of agglutination of insoluble carrier particlesusing samples with the known concentration of the antigenic substances.In this measurement, for example, the less agglutination is formed, themore transparent the reaction mixture is. On the other hand, in case ofmeasuring an agglutination quantitatively, for example, an opticalmeasurement of agglutination is preferable in the light of easiness ofmeasurement. As a method for an optical measurement of agglutination ofinsoluble carrier particles such as latex, the known methods for anoptical measurement can be used. Examples include various methods suchas nephelometry wherein formation of an agglutination is correlated toan increase of turbidity; a method of measuring a distribution ofparticles size wherein formation of an agglutination is correlated to achange in a distribution of particle size or an average diameter ofparticles; an integrating sphere turbidimetric assay wherein formationof an agglutination is correlated to a ratio of intensity of scatteringlight measured with an integrating sphere to intensity of transmittedlight. A rate assay or an end-point assay can be applied to each ofthese methods for measurement. In the rate assay, degrees ofagglutination are determined by an agglutination reaction rate, which iscalculated on the basis of the differences of values to be measured atleast two different points of the reaction. In the end-point assay,degrees of agglutination are determined by the values to be determinedat a specific point, a point usually considered to be an end-point ofthe reaction. In the light of easiness and rapidness of measurement, arate assay using nephelometry is preferable.

[0029] In the optical measurement of an agglutination of insolublecarrier particles such as latex with an average diameter of 0.04 to 0.8μm, measurement by using a light with a wavelength of about 400 to 1400nm is preferable.

[0030] As a reagent for an immunoassay according to the presentinvention, there is no specific limitation to a composition of thereagent as long as the reagent comprises a reagent for a measurement ofan antigenic substance to be determined and a compound having a grouprepresented by formula (I) such as a phoshorylchlorine group. Examplesinclude a reagent kit comprising insoluble carrier particles, buffer,antibodies that bind to an antigen in a sample, and a compound having agroup represented by formula (I), and the reagent kit may, if necessary,comprise surfactant, antiseptic, antibodies that bind to the antibodiesto the antigen in a sample, and the like. In particular, a specificexample of a reagent kit includes a reagent kit comprising a firstreagent and a second reagent: a first reagent comprising insolublecarrier particles and buffer; and a second reagent comprising a compoundhaving a group represented by formula (I) and antibodies that bind tothe antigen. The first reagent and the second reagent in the reagent kitmay further comprise, if necessary, surfactant, antiseptic, antibodythat binds to the antigen in the sample, antibodies that bind to theantigen in a sample, antibodies for formation of an antibody complexthat bind to the antibodies to the antigen in a sample, and the like.

EXAMPLES

[0031] The present invention will be described further in detail by thefollowing Examples, Comparative Examples and Reference Examples, whilethe technical scope of the present invention will not be limited tothese examples and the like. Reagents of the following composition wereprepared. Example 1 [Reagent R1] HEPES buffer (manufactured by DOJINDOLaboratories, pH 7.8) 4.77 g/L Latex particles (particle diameter:0.0775 μm, manufactured by 0.033% by weight/L SEKISUI Chemical Co. Ltd.)NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/L [Reagent R2]HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.0) 4.77 g/LSodium chloride (manufactured by Wako Pure Chemical 15 g/L Industries,Ltd.) Polymer 2 (prepared in Reference Example 2) 2 g/L NaN₃(manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Anti-human HbA1cmouse monoclonal antibody (preparation in 0.025 g (in terms of IgG)/LPreparation Example 1) Anti-mouse IgG goat polyclonal antibody(manufactured by Wako 0.025 g (in terms of IgG)/L Pure ChemicalIndustries, Ltd.) Example 2 [Reagent R1] HEPES buffer (manufactured byDOJINDO Laboratories, pH 7.8) 4.77 g/L Latex particles (particlediameter: 0.0775 μm, manufactured by 0.033% by weight/L SEKISUI ChemicalCo. Ltd.) NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/L[Reagent R2] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.0)4.77 g/L Sodium chloride (manufactured by Wako Pure Chemical 15 g/LIndustries, Ltd.) Polymer 3 (prepared in Reference Example 3) 2 g/L NaN₃(manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Anti-human HbA1cmouse monoclonal antibody 0.025 g (in terms of IgG)/L

[0032] Anti-mouse IgG goat polyclonal antibody (manufactured by WakoPure Chemical Industries, Ltd.) 0.025 g (in terms of IgG)/L

Comparative Example 1

[0033] Reagents of the following composition comprising Tween 20 insteadof polymer 2 in Example 1 or polymer 3 in Example 2 were prepared.[Reagent R1] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8)4.77 g/L Latex particles (particle diameter: 0.0775 μm, manufactured by0.033% by weight/L SEKISUI Chemical Co. Ltd.) NaN₃ (manufactured byKanto Chemical Co., Inc.) 0.1 g/L [Reagent R2] HEPES buffer(manufactured by DOJINDO Laboratories, pH 7.0) 4.77 g/L Sodium chloride(manufactured by Wako Pure Chemical 15 g/L Industries, Ltd.) Tween 20(manufactured by Wako Pure Chemical Industries, Ltd.) 2 g/L NaN₃(manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Anti-human HbA1cmouse monoclonal antibody 0.025 g (in terms of IgG)/L

[0034] Anti-mouse IgG goat polyclonal antibody (manufactured by WakoPure Chemical Industries, Ltd.) 0.025 g (in terms of IgG)/L

Comparative Example 2

[0035] Reagents of the following composition comprising Brij 30 insteadof polymer 2 in Example 1 or polymer 3 in Example 2 were prepared.[Reagent R1] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8)4.77 g/L Latex particles (particle diameter: 0.0775 μm, manufactured by0.033% by weight/L SEKISUI Chemical Co. Ltd.) NaN₃ (Kanto Chemical Co.,Inc.) 0.1 g/L [Reagent R2] HEPES buffer (manufactured by DOJINDOLaboratories, pH 7.0) 4.77 g/L Sodium chloride (manufactured by WakoPure Chemical Industries, 15 g/L Ltd.) Brij 30 (manufactured by Sigma) 2g/L NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Anti-humanHbA1c mouse monoclonal antibody 0.025 g (in terms of IgG)/L

[0036] Anti-mouse IgG goat polyclonal antibody (manufactured by WakoPure Chemical Industries, Ltd.) 0.025 g (in terms of IgG)/L

Comparative Example 3

[0037] Reagents of the following composition comprising Brij 56 insteadof polymer 2 in Example 1 or polymer 3 in Example 2 were prepared.[Reagent R1] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8)4.77 g/L Latex particles (particle diameter: 0.0775 μm, manufactured by0.033% by weight/L SEKISUI Chemical Co. Ltd.) NaN₃ (manufactured byKanto Chemical Co., Inc.) 0.1 g/L [Reagent R2] HEPES buffer(manufactured by DOJINDO Laboratories, pH 7.0) 4.77 g/L Sodium chloride(manufactured by Wako Pure Chemical Industries, 15 g/L Ltd.) Brij 56(manufactured by Sigma) 2 g/L NaN₃ (manufactured by Kanto Chemical Co.,Inc.) 0.1 g/L Anti-human HbA1c mouse monoclonal antibody 0.025 g (interms of IgG)/L Anti-mouse IgG goat polyclonal antibody (manufactured byWako 0.025 g (in terms of IgG)/L Pure Chemical Industries, Ltd.)

Example 3

[0038] Human blood was collected by EDTA blood collecting tube (VENOJECTGlass Vacuum Tubes; TERUMO Corp.) and left to stand for two hours. Theprepicitated hemocyte layer (10 μL) was taken and the hemocyte layer wasdiluted with purified water (1 mL). The diluted hemocyte layer wasfrozen and stored at −20. degree. C., and the frozen hemocyte layer wasmelted just before use to give a sample. Determination of theconcentration of HbA1c was carried out by using Reagent R1 and ReagentR2 prepared in Example 1, Example 2, Comparative Example 1, ComparativeExample 2 and Comparative Example 3, respectively. Further, thecalibration curve was diagramed by using the reagent just afterunsealing, along with each of standard samples with HbA1c values of0.0%, 4.2%, 7.7%, 11.3%, 14.8%, which were determined by glycohemoglobinautoanalyzer HLC-723 GHbV (Tosoh Corporation) using the reagent justafter unsealing. Determination of HbA1c in the sample was carried out asfollows. The sample (8 μL) prepared as above was added to Reagent R1(240 μL), and the reaction was allowed to proceed at 37. degree. C. for5 minutes. Then, Reagent R2 (80 μL) was added thereto, and the reactionwas allowed to proceed at 37. degree. C. for 5 minutes. The change inabsorbance was measured by the 2 point-end method (photometric points:16-34) by Hitachi autoanalyzer 7170 at the main-wavelength of 450 nm andthe sub-wavelength of 800 nm. Finally, the change in absorbance wascorrelated to the concentration of HbA1c in the sample on the diagram.This operation was repeated ten times to determine the average, thestandard deviation and the within-run reproducibility [(the standarddeviation×100)/the average)]. The results are shown in Table 1. As shownin Table 1, in comparison with Comparative Examples 1 to 3, degrees ofagglutination were almost constant to give improved reproducibility inExamples 1 and 2. TABLE 1 Number of Compar- Compar- Compar- times ofExam- ative ative ative determination ple 1 Example 2 Example 1 Example2 Example 3  1 6.3 6.3 6.3 6.3 6.5  2 6.4 6.3 6.4 6.4 6.4  3 6.3 6.3 6.36.3 6.1  4 6.3 6.3 6.4 6.2 6.3  5 6.3 6.3 6.3 6.6 6.2  6 6.4 6.4 6.5 6.56.4  7 6.3 6.3 6.2 6.2 6.5  8 6.3 6.3 6.2 6.2 6.3  9 6.3 6.3 6.3 6.3 6.410 6.3 6.3 6.4 6.2 6.4 average 6.3 6.3 6.3 6.3 6.4 standard 0.040 0.0300.090 0.133 0.120 deviation within-run 0.63% 0.48% 1.42% 2.10% 1.90%reproducibility

Example 4

[0039] Reagents of the following composition were prepared. [Reagent R1]HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8) 4.77 g/LLatex particles (particle diameter: 0.0775 μm, manufactured by 0.033% byweight/L SEKISUI Chemical Co. Ltd.) NaN₃ (manufactured by Kanto ChemicalCo., Inc.) 0.1 g/L Polymer 4 (prepared in Reference Example 4) 1 g/L[Reagent R2] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.0)4.77 g/L Sodium chloride (manufactured by Wako Pure Chemical Industries,15 g/L Ltd.) Tween 20 (manufactured by Wako Pure Chemical Industries,Ltd.) 2 g/L NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/LAnti-human HbA1c mouse monoclonal antibody 0.025 g (in terms of IgG)/LAnti-mouse IgG goat polyclonal antibody (manufactured by Wako 0.025 g(in terms of IgG)/L Pure Chemical Industries, Ltd.)

Example 5

[0040] Reagents of the following composition were prepared. [Reagent R1]HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8) 4.77 g/LLatex particles (particle diameter: 0.0775 μm, manufactured by 0.033% byweight/L SEKISUI Chemical Co. Ltd.) NaN₃ (manufactured by Kanto ChemicalCo., Inc.) 0.1 g/L Polymer 5 (prepared in Reference Example 5) 1 g/L[Reagent R2] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.0)4.77 g/L Sodium chloride (manufactured by Wako Pure Chemical Industries,15 g/L Ltd.) Tween 20 (manufactured by Wako Pure Chemical Industries,Ltd.) 2 g/L NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/LAnti-human HbA1c mouse monoclonal antibody 0.025 g (in terms of IgG)/LAnti-mouse IgG goat polyclonal antibody (manufactured by Wako 0.025 g(in terms of IgG)/L Pure Chemical Industries, Ltd.)

[0041] Example 6 Reagents of the following composition were prepared.[Reagent R1] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8)4.77 g/L Latex particles (particle diameter: 0.0775 μm, manufactured by0.033% by weight/L SEKISUI Chemical Co. Ltd.) NaN₃ (manufactured byKanto Chemical Co., Inc.) 0.1 g/L Polymer 1 (prepared in ComparativeExample 1) 0.002 g/L [Reagent R2] HEPES buffer (manufactured by DOJINDOLaboratories, pH 7.0) 4.77 g/L Sodium chloride (manufactured by WakoPure Chemical Industries, 15 g/L Ltd.) Tween 20 (manufactured by WakoPure Chemical Industries, Ltd.) 2 g/L NaN₃ (manufactured by KantoChemical Co., Inc.) 0.1 g/L Anti-human HbA1c mouse monoclonal antibody0.025 g (in terms of IgG)/L Anti-mouse IgG goat polyclonal antibody(manufactured by Wako 0.025 g (in terms of IgG)/L Pure ChemicalIndustries, Ltd.) Comparative Example 4 Reagents of the followingcomposition which do not comprise polymer 4 in Example 4, polymer 5 inExample 5 and polymer 1 in Example 6 were prepared. [Reagent R1] HEPESbuffer (manufactured by DOJINDO Laboratories, pH 7.8) 4.77 g/L Latexparticles (particle diameter: 0.0775 μm, manufactured by 0.033% byweight/L SEKISUI Chemical Co. Ltd.) NaN₃ (manufactured by Kanto ChemicalCo., Inc.) 0.1 g/L [Reagent R2] HEPES buffer (manufactured by DOJINDOLaboratories, pH 7.0) 4.77 g/L Sodium chloride (manufactured by WakoPure Chemical Industries, 15 g/L Ltd.) Tween 20 (manufactured by WakoPure Chemical Industries, Ltd.) 2 g/L NaN₃ (manufactured by KantoChemical Co., Inc.) 0.1 g/L Anti-human HbA1c mouse monoclonal antibody0.025 g (in terms of IgG)/L Anti-mouse IgG goat polyclonal antibody(manufactured by Wako 0.025 g (in terms of IgG)/L Pure ChemicalIndustries, Ltd.)

Example 7

[0042] Human blood was collected by EDTA blood collecting tube (VENOJECTGlass Vacuum Tubes; TERUMO Corp.) and left to stand for two hours. Theprepicitated hemocyte layer (10 μL) was taken and the hemocyte layer wasdiluted with purified water (1 mL). The diluted hemocyte layer wasfrozen and stored at −20. degree. C., and the frozen hemocyte layer wasmelted just before use to give a sample. Determination of theconcentration of HbA1c was carried out in the same manner as in Example3, by using the sample along with Reagent R1 and Reagent R2 prepared inExamples 4 to 6, and Comparative Example 4, respectively. The resultsare shown in Table 2. As shown in Table 2, in Examples 4 to 6, degreesof agglutination were almost constant to give improved reproducibilityin comparison with Comparative Example 4. TABLE 2 Number of timesComparative of determination Example 4 Example 5 Example 6 Example 4  16.3 6.3 6.3 6.5  2 6.3 6.3 6.4 6.3  3 6.3 6.2 6.2 6.3  4 6.3 6.3 6.3 6.1 5 6.3 6.3 6.2 6.2  6 6.3 6.2 6.3 6.4  7 6.3 6.3 6.3 6.3  8 6.3 6.3 6.36.4  9 6.2 6.3 6.3 6.3 10 6.3 6.3 6.3 6.2 average 6.3 6.3 6.3 6.3standard deviation 0.030 0.040 0.054 0.110 within-run 0.48% 0.64% 0.86%1.74% reproducibility

Example 8

[0043] Reagents of the following composition were prepared. [Preparationof R1 solution (latex)] HEPES buffer (manufactured by DOJINDOLaboratories, pH 7.8) 4.77 g/L Latex (particle diameter: 0.0775 μm,manufactured by SEKISUI 0.033% by weight/L Chemical Co. Ltd.) NaN₃(manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Polymer 4 (preparedin Reference Example 4) 2 g/L [Preparation of R2 (antibody) solution]HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.0) 4.77 g/LSodium chloride (manufactured by Wako Pure Chemical Industries, 15 g/LLtd.) Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) 2g/L NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Anti-humanHbA1c mouse monoclonal antibody 0.025 g (in terms of IgG)/L Anti-mouseIgG goat polyclonal antibody (manufactured by Wako 0.025 g (in terms ofTgG)/L Pure Chemical Industries, Ltd.)

Example 9

[0044] Reagents of the following composition were prepared. [Reagent R1]HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.8) 4.77 g/LLatex (particle diameter: 0.0775 μm, manufactured by SEKISUI 0.033% byweight/L Chemical Co. Ltd.) NaN₃ (manufactured by Kanto Chemical Co.,Inc.) 0.1 g/L Polymer 4 (prepared in Reference Example 4) 5 g/L [ReagentR2] HEPES buffer (manufactured by DOJINDO Laboratories, pH 7.0) 4.77 g/LSodium chloride (manufactured by Wako Pure Chemical Industries, 15 g/LLtd.) Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.) 2g/L NaN₃ (manufactured by Kanto Chemical Co., Inc.) 0.1 g/L Anti-humanHbA1c mouse monoclonal antibody 0.025 g (in terms of IgG)/L Anti-mouseIgG goat polyclonal antibody (manufactured by Wako 0.025 g (in terms ofIgG)/L Pure Chemical Industries, Ltd.)

Example 10

[0045] Human blood was collected by EDTA blood collecting tube (VENOJECTGlass Vacuum Tubes; TERUMO Corp.) and left to stand for two hours. Theprepicitated hemocyte layer (10 μL) was taken, and the hemocyte layerwas made to 1 mL with purified water to give a sample 1. Further, humanblood was collected by EDTA blood collecting tube (VENOJECT Glass VacuumTubes; TERUMO Corp.) and left to stand for two hours. A supernatant (4μL) and a prepicitated hemocyte layer (10 μL) were taken, and thesupernatant and the prepicitated hemocyte layer were made to 1 mL withpurified water to give a sample 2. Determination of the concentration ofHbA1c in the samples was carried out in the same manner as in Example 3by using these samples along with Reagent R1 and Reagent R2 prepared inExample 4, Example 8, Example 9 and Comparative Example 4, respectively.Averages in three times of determinations for each sample are shown inTable 3. As shown in Table 3, in Example 4, Example 8 and Example 9,particularly, in Examples 8 and 9 with higher content of polymer 4,thedegrees of agglutination were almost constant to give improvedreproducibility even in the determination using the sample 2 comprisingplasma, in comparison with Comparative Example 4. TABLE 3 Reagent Sample1 Sample 2 Example 4 6.6 6.1 Example 8 6.7 6.5 Example 9 6.7 6.7Comparative 6.6 5.1 Example 4

Reference Example 1 (Preparation of Polymer 1)

[0046] MPC (NOF Corporation) (35.7 g) and BMA (Wako Pure ChemicalIndustries, Inc.) (4.3 g), dissolved in ethanol (160 g), were put into afour-neck flask, and nitrogen gas was bubbled into the ethanol solutionfor 30 minutes. After heating the ethanol solution to 60. degree. C.,polymerization initiator AIBN (Wako Pure Chemical Industries, Inc.)(0.82 g) was added thereto, and the polymerization reaction was allowedto proceed for 8 hours. With stirring the solution after thepolymerization reaction, the solution was added dropwise to diethylether (3 L). The formed precipitate was filtered, and dried in vacuo atroom temperature for 48 hours to give the copolymer (polymer 1) as apowder with the ratio of MPC (0.8 mol) to BMA (0.2 mol). Molecularweight of this polymer 1 was evaluated 153,000 as a weight-averagemolecular weight, by gel permeation chromatography (abbreviatedhereinafter as GPC) analysis. The GPC analysis was carried out under thefollowing condition: eluent: phosphate buffer (pH 7.4, 20 mM); standard:polyethyleneglycol; detection: UV (210 nm) and refractive index (thiscondition in GPC analysis was also used in References 2 to 5).

Reference Example 2 (Preparation of polymer 2)

[0047] MPC (20.3 g) and BMA (9.75 g), dissolved in ethanol (120 g), wereput into a four-neck flask, and nitrogen gas was bubbled into theethanol solution for 30 minutes. After heating the ethanol solution to60. degree. C., AIBN (0.35 g) was added thereto, and the polymerizationreaction was allowed to proceed for 8 hours. With stirring the solutionafter the polymerization reaction, the solution was added dropwise todiethyl ether (3 L). The formed precipitate was filtered, and dried invacuo at room temperature for 48 hours to give the copolymer (polymer 2)as a powder with the ratio of MPC (0.5 mol) to BMA (0.5 mol). Molecularweight of this polymer 2 was evaluated 224,000 as a weight-averagemolecular weight, by GPC analysis.

Reference Example 3 (Preparation of polymer 3)

[0048] MPC (14.1 g) and BMA (15.9 g), dissolved in ethanol (120 g), wereput into a four-neck flask, and nitrogen gas was bubbled into theethanol solution for 30 minutes. After heating the ethanol solution to60. degree. C., AIBN (0.35 g) was added thereto, and the polymerizationreaction was allowed to proceed for 8 hours. With stirring the solutionafter the polymerization reaction, the solution was added dropwise todiethyl ether (3 L). The formed precipitate was filtered, and dried invacuo at room temperature for 48 hours to give the copolymer (polymer 3)as a powder with the ratio of MPC (0.3 mol) to BMA (0.7 mol). Molecularweight of this polymer 3 was evaluated 130,000 as a weight-averagemolecular weight, by. GPC analysis.

Reference Example 4 (Preparation of polymer 4)

[0049] MPC (50.0 g), dissolved in ethanol (160 g), was put into afour-neck flask, and nitrogen gas was bubbled into the ethanol solutionfor 30 minutes. After heating the ethanol solution to 60. degree. C.,AIBN (0.24 g) was added thereto, and the polymerization reaction wasallowed to proceed for 8 hours. With stirring the solution after thepolymerization reaction, the solution was added dropwise to diethylether (3 L). The formed precipitate was filtered, and dried in vacuo atroom temperature for 48 hours to give the homopolymer of MPC (polymer 4)as a powder. Molecular weight of this polymer 4 was evaluated 529,000 asa weight-average molecular weight, by GPC analysis.

Reference Example 5 (Preparation of polymer 5)

[0050] MPC (30.0 g), dissolved in ethanol (120 g), was put into afour-neck flask, and nitrogen gas was bubbled into the ethanol solutionfor 30 minutes. After heating the ethanol solution to 60. degree. C.,AIBN (0.48 g) was added thereto, and the polymerization reaction wasallowed to proceed for 8 hours. With stirring the solution after thepolymerization reaction, the solution was added dropwise to diethylether (3 L). The formed precipitate was filtered, and dried in vacuo atroom temperature for 48 hours to give the homopolymer of MPC (polymer 5)as a powder. Molecular weight of this polymer 5 was evaluated 183,000 asa weight-average molecular weight, by GPC analysis. Reference Example 6(Preparation of the monoclonal antibody to the glycopeptide epitope ofthe N terminus of the β chain in hemoglobin A1c)

[0051] According to the description of the Preparation Example 1 inJapanese Published Unexamined Patent Application No. 35752/95, thepeptide of Val-His-Leu-Thr-Pro-Cys, corresponding to the amino acidsequence of the N terminus of the β chain in hemoglobin, was prepared,and glucose was allowed to bind non-enzymatically to the a-amino groupof Val, the amino acid residue of the N terminus to give theglycopeptide. Further, the glycopeptide was allowed to bind to thecarrier protein through a spacer to give the immunogen. The monoclonalantibody that reacts to HbA1c but does not react to HbA0 was obtained byconventional method. The obtained monoclonal antibody was an antibodywhich does not react to free HbA1c, but reacts to HbA1 bound toinsoluble carrier particles.

Industrial Applicability

[0052] According to the present invention wherein a compound having agroup represented by formula (I) such as a phosphorylcholine group isused, a stable and uniform reaction of immunological agglutination usinginsoluble carrier particles such as latex proceeds to give adetermination with good reproducibility.

1. An agglutination immunoassay which comprises allowing an antigenicsubstance in a sample to bind to insoluble carrier particles carryingsubstantially neither antigens nor antibodies thereon, and allowing anantibody or an antibody complex which reacts specifically to theantigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles, wherein acompound having a group represented by formula (I),

wherein n is an integer of 1 to 6, and R¹, R² and R³ are the same ordifferent, and independently represent hydrogen, substituted orunsubsitituted alkyl having 1 to 6 carbon atoms, is used.
 2. Theagglutination immunoassay according to claim 1, wherein the compoundhaving a group represented by formula (I) is a compound prepared bypolymerization of a monomer having a group represented by formula (I).3. The agglutination immunoassay according to claim 1, wherein thecompound having a group represented by formula (I) is a compoundprepared by polymerization of a monomer having a group represented byformula (I) with other monomer polymerizable with the monomer having agroup represented by formula (I).
 4. The agglutination immunoassayaccording to claim 3, wherein the other monomer polymerizable with themonomer having a group represented by formula (I) is a monomer having avinyl group.
 5. The agglutination immunoassay according to claim 4,wherein the monomer having a vinyl group is n-butyl methacrylate.
 6. Theagglutination immunoassay according to any of claims 2 to 5, wherein themonomer having a group represented by formula (I) is a monomer having agroup represented by formula (I) and a vinyl group.
 7. The agglutinationimmunoassay according to any of claims 1 to 6, wherein the grouprepresented by formula (I) is a phosphorylcholine group.
 8. Theagglutination immunoassay according to claim 6, wherein the monomerhaving a group represented by formula (I) and a vinyl group is2-methacryloyloxyethyl phosphorylcholine.
 9. The agglutinationimmunoassay according to any of claims 1 to 8, wherein the antibody isan anti-hemoglobin A1c monoclonal antibody.
 10. The agglutinationimmunoassay according to any of claims 1 to 9, wherein the antibodycomplex comprises an antibody which reacts specifically to the antigenicsubstance, and a secondary antibody which reacts selectively to theantibody which reacts specifically to the antigenic substance.
 11. Theagglutination immunoassay according to any of claims 1 to 10, whereinthe insoluble carrier particles are polystyrene latex.
 12. A reagent foran immunoassay comprising a compound having a group represented byformula (I),

wherein n is an integer of 1 to 6, and R¹, R² and R³are the same ordifferent, and independently represent hydrogen, substituted orunsubsitituted alkyl having 1 to 6 carbon atoms, used in anagglutination immunoassay which comprises allowing an antigenicsubstance in a sample to bind to insoluble carrier particles carryingsubstantially neither antigens nor antibodies thereon, and allowing anantibody or an antibody complex which reacts specifically to theantigenic substance to bind to the antigenic substance to give aselective agglutination of the insoluble carrier particles.
 13. Thereagent for an immunoassay according to claim 12, wherein the compoundhaving a group represented by formula (I) is a compound prepared bypolymerization of a monomer having a group represented by formula (I).14. The reagent for an immunoassay according to claim 12, wherein thecompound having a group represented by formula (I) is a compoundprepared by polymerization of a monomer having a group represented byformula (I) with other monomer polymerizable with the monomer having agroup represented by formula (I).
 15. The reagent for an immunoassayaccording to claim 14, wherein the other monomer polymerizable with themonomer having a group represented by formula (I) is a monomer having avinyl group.
 16. The reagent for an immunoassay according to claim 15,wherein the monomer having a vinyl group is n-butyl methacrylate. 17.The reagent for an immunoassay according to any of claims 13 to 16,wherein the monomer having a group represented by formula (I) is amonomer having a group represented by formula (I) and a vinyl group. 18.The reagent for an immunoassay according to any of claims 12 to 17,wherein the group represented by formula (I) is a phosphorylcholinegroup.
 19. The reagent for an immunoassay according to claim 17, whereinthe monomer having a group represented by formula (I) and a vinyl groupis 2-methacryloyloxyehtyl phosphorylcholine.
 20. The reagent for animmunoassay according to any of claims 12 to 19, wherein the antibody isan anti-hemoglobin A1c monoclonal antibody.
 21. The reagent for animmunoassay according to any of claims 12 to 20, wherein the antibodycomplex comprises an antibody which reacts specifically to the antigenicsubstance, and a secondary antibody which reacts selectively to theantibody which reacts specifically to the antigenic substance.
 22. Thereagent for an immunoassay according to any of claims 12 to 21, whereinthe insoluble carrier particles are polystyrene latex.